During the 2009 fiscal year, we accomplished the following: 1. Developed conditions to use 10-12kb DNA probes for fluorescent in-situ hybridization (FISH) experiments. 2. Validated nuclear positioning and chromosome compaction results using an entirely different procedure that does not require heat denaturation of chromosomes to permit probe hybridization. 3. Utilized anti-CTCF and anti-cohesin antibodies for immunohistochemical analysis of pro-B cells. We observed a punctate staining pattern in interphase cells, indicating sub-nuclear domains enriched in these proteins. We co-localized the IgH locus to CTCF-enriched foci by laser confocal microscopy and immuno-FISH. 4. We generated Tet-inducible cell lines that down-regulate CTCF after doxicycline treatment. In these cells, CTCF foci were lost and replaced by diffuse staining.